The natural compound gracillin exerts potent antitumor activity by targeting mitochondrial complex II.

Mitochondria play a pivotal role in cancer bioenergetics and are considered a potential target for anticancer therapy. Considering the limited efficacy and toxicity of currently available mitochondria-targeting agents, it is necessary to develop effective mitochondria-targeting anticancer drugs. By screening a large chemical library consisting of natural products with diverse chemical entities, we identified gracillin, a steroidal saponin, as a mitochondria-targeting antitumor drug. Gracillin displayed broad-spectrum inhibitory effects on the viability of a large panel of human cancer cell lines, including those carrying acquired resistance to chemotherapy or EGFR-targeting drugs, by inducing apoptosis. We show that gracillin attenuates mitochondria-mediated cellular bioenergetics by suppressing ATP synthesis and by producing reactive oxygen species (ROS). Mechanistically, gracillin disrupts complex II (CII) function by abrogating succinate dehydrogenase (SDH) activity without affecting the succinate:ubiquinone reductase. The gracillin-induced cell death was potentiated by 3-nitropropionic acid (3-NPA) or thenoyltrifluoroacetone (TTFA), which inhibit CII by binding to the active site of SDHA or to the ubiquinone-binding site, respectively. Finally, we show that gracillin effectively suppressed the mutant-Kras-driven lung tumorigenesis and the growth of xenograft tumors derived from cell lines or patient tissues. Gracillin displayed no obvious pathophysiological features in mice. Collectively, gracillin has potential as a CII-targeting antitumor drug.

Esterase inhibitors as ester-containing drug stabilizers and their hydrolytic products: potential contributors to the matrix effects on bioanalysis by liquid chromatography/tandem mass spectrometry.

RATIONALE: Esterase inhibitors are widely used to stabilize ester-containing drugs in biological matrices for quantitative liquid chromatography/tandem mass spectrometry (LC/MS/MS) assays. These co-existing inhibitors could cause matrix effects on bioanalysis and jeopardize the assay performance. We therefore developed an LC/MS/MS methodology to monitor the fate of inhibitors and evaluate their matrix effects, which is described in this study. METHODS: Human plasma containing 20 mM of diisopropylfluorophosphate (DFP), paraoxon, eserine, phenylmethylsulfonyl fluoride (PMSF) or 2-thenoyltrifluoroacetone (TTFA) was extracted by liquid-liquid extraction (LLE) and analyzed by an LC/MS/MS assay for BMS-068645 (a model drug) with additional pre-optimized selected reaction monitoring (SRM) transitions using positive/negative electrospray ionization (ESI) mode for each inhibitor. Hydrolytic products were characterized by product ion or neutral loss scan LC/MS/MS analysis. The matrix effect contribution from each inhibitor was evaluated by post-column infusion of BMS-068645. RESULTS: In the extracted samples by LLE, SRM chromatograms revealed the presence of paraoxon, eserine and TTFA with peak intensity of >2.50E08. Three DFP hydrolytic products, diisopropyl phosphate (DP), triisopropyl phosphate (TP) and DP dimer, and one PMSF hydrolytic product, phenymethanesulfonic acid (PMSA), were identified in the extracted samples. In post-column infusion profiles, ion suppression or enhancement was observed in the retention time regions of eserine (~10% suppression), paraoxon (~70% enhancement) and DP dimer (~20% suppression). CONCLUSIONS: The SRM transitions described here make it possible to directly monitor the inhibitors and their hydrolytic products. In combination with post-column infusion, this methodology provides a powerful tool to routinely monitor the matrix effects-causing inhibitors, so that their matrix effects on the bioanalysis can be evaluated and minimized.

Characterization of the respiratory chain of Helicobacter pylori.

The respiratory chain of Helicobacter pylori has been investigated. The total insensitivity of activities of NADH dehydrogenase to rotenone and of NADH-cytochrome c reductase to antimycin is indicative of the absence of the classical complex I of the electron transfer chain in this bacterium. NADPH-dependent respiration was significantly stronger than NADH-dependent respiration, indicating that this is a major respiratory electron donor in H. pylori. Fumarate and malonate exhibited a concentration-dependent inhibitory effect on the activity of succinate dehydrogenase. The activity of succinate-cytochrome c reductase was inhibited by antimycin, implying the presence of a classical pathway from complex II to complex III in this bacterium. The presence of NADH-fumarate reductase (FRD) was demonstrated in H. pylori and fumarate could reduce H2O2 production from NADH, indicating fumarate to be an endogenous substrate for accepting electrons from NADH. The activity of NADH-FRD was inhibited by 2-thenoyltrifluoroacetone. A tentative scheme for the electron transfer pathway in H. pylori is proposed, which may be helpful in clarifying the pathogenesis of H. pylori and in opening new lines for chemotherapy against this bacterium.

The probable site of action of thenolytrifluoracetone on the respiratory chain.

1. It is shown that the electron-transfer inhibitor thenoyltrifluoroacetone abolishes a respiratory-chain electron-paramagnetic-resonance absorbance due to spin-spin interactions of ubisemiquinones at concentrations similar to those required for inhibition of succinate oxidation. 2. A specific site of interaction of thenoyltrifluoroacetone with the respiratory chain is proposed to be on the ubisemiquinone with which succinate dehydrogenase reacts. 3. Our results further demonstrate the close association of the HiPIP (high-potential iron-sulphur) centre of succinate dehydrogenase with ubisemiquinone.